Producing potent high-affinity antibodies that can neutralize target pathogens remains a central goal for vaccine research. While a great deal is known about antibody molecules themselves, still too little is understood about the programming of high-affinity memory B cells that produce them. Many promising vaccine antigens fail to elicit protective antibodies using contemporary vaccine formulations. Thus, major gaps remain in our basic understanding of antibody affinity maturation and how to enhance poor immunogenicity using rational protein vaccination. Research Focus: Adjuvants are required to induce potent antibody responses using protein-based vaccine formulations. Follicular helper T (TFH) cells are a new class of immune regulator specialized to control multiple stages of memory B cell development. Here, we examine the innate cellular and molecular regulators of antigen-specific TFH development at the point of initial priming and following the vaccine boost. Specific Aims: Using new single cell strategies, we will dissect the molecular components of regulatory programs within peptide MHCII-expressing dendritic cell (DC) that control antigen-specific TFH programming in vivo (SA-1). Upon antigen recall, we now reveal that memory B cells form secondary GC reactions, re-diversify their expressed BCR, with evidence for ongoing clonal selection. Importantly, addition of secondary adjuvant at the vaccine boost enhanced affinity maturation within the memory B cell response. These recent findings alter the prevailing view of BCR affinity maturation and indicate new innate mechanisms for regulating high-affinity B cell memory (SA-2). Impact: We use polyclonal murine models of protein vaccination with state-of-the-art single cell analyses of antigen-specific immune function. These high-resolution studies of model antigens have the capacity to shift the basic conceptual framework that surrounds existing vaccine paradigms. Importantly, these basic new principles and assays can be used to re-design contemporary vaccine formulations that optimize high-affinity B cell immunity to more complex antigens. PUBLIC HEALTH RELEVANCE: Vaccines rely on antibodies to protect us for long periods against infection. We know a lot about how antibody molecules function but very little about the memory cells that produce them. In order to design better vaccines, we need to understand how the vaccine components control the memory cells at the first vaccination and at the vaccine boost.